Method for synthesis of nitroxyl radical

ABSTRACT

The problem to be solved by the present invention is to provide a highly-versatile method for producing a nitroxyl radical derivative, in which position-2 and position-6 in a TEMPO-based compound can be easily substituted, and further, a method for producing a nitroxyl radical derivative, in which a nitrogen nucleus is labeled with  15 N. The above-described problem can be solved by reacting a triacetoneamine derivative with ketone or aldehyde in the presence of ammonium salt or a  15 N-labeled compound thereof to obtain a 2,6-substituted-4-piperidone derivative.

TECHNICAL FIELD

The present invention relates to a method for producing a nitroxylradical derivative, in particular, a nitroxyl radical derivative havingsubstituents at position-2 and position-6 in a TEMPO-based compound.

BACKGROUND ART

Nitroxyl radical is a substance having an unpaired electron, and becauseof a variety of properties thereof, it is widely used as anantioxidative substance, a chemical cell, a polymerization agent or thelike. In addition, nitroxyl radical is highly sensitive to a freeradical such as active oxygen, and the distribution of nitroxyl radicalin vivo varies depending on the basic structure thereof and the type ofsubstituent. Therefore, utilizing these natures, nitroxyl radical canalso be used as a contrast agent for following a free radical reactionin vivo. The present inventors focused their attention on this point andalready indicated that novel image analysis can be carried out in vivoby utilizing the simultaneous separate imaging method using ¹⁴N-labeledand ¹⁵N-labeled compounds (see H. Utsumi, K. Yamada, K. Ichikawa, K.Sakai, Y Kinoshita, S. Matsumoto and M. Nagai, PNAS, 103, 1463 (2006)).

Conventionally, nitroxyl radical is generally synthesized from acetone,ammonia (or ammonia chloride), etc. Typical examples of nitroxylradicals obtained using this technique include2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) derivatives and2,2,5,5-tetramethylpyrrolidine-N-oxyl (PROXYL) derivatives.

Reactivities of these conventional nitroxyl radicals to a free radicalsignificantly differ from each other depending on the difference ofskeleton (TEMPO-based or PROXYL-based). However, reactivities ofTEMPO-based compounds do not differ from each other so much. It isthought that this is because structures around the unpaired electron innitroxyl radical, i.e., substituents at position-2 and position-6 arenot substituted with other substituents.

Several research groups have attempted to carry out replacement ofsubstituents at position-2 and position-6 in a TEMPO-based compound. Forexample, Miura et al. succeeded in synthesis of2,6-dispirocyclohexane-4-piperidone using ammonia as a starting materialand acetonine as an intermediate (see Y. Miura, N. Nakamura and I.Taniguchi, Macromolecules, 34, 447 (2001)). Wetter et al. succeeded insynthesis of 2,2,6,6-tetraethyl-4-oxo-TEMPO using bisphosphonate as astarting material (see C. Wetter, J. Gierlich, C. A. Knoop, C. Müller,T. Schulte and A. Studer, Chem. Eur. J., 10, 1156 (2004)).

However, these synthesis methods have the following problems: thestability of an intermediate compound such as acetonine is low; manysteps for synthesis are required; and there is a lack of application tosynthesis of other compounds; etc.

DISCLOSURE OF THE INVENTION

Under the above-described circumstances, a highly-versatile method forproducing a nitroxyl radical derivative, in which substituents can beintroduced into position-2 and position-6 in a TEMPO-based compoundusing a simple method, and further, a method for producing a nitroxylradical derivative, in which a nitrogen nucleus is labeled with ¹⁵N, aredesired.

The present inventors diligently made researches in order to solve theabove-described problems, and found that substituents can be easilyintroduced into position-2 and position-6 in a triacetoneaminederivative by reacting the triacetoneamine derivative as a startingmaterial with ketone or aldehyde in the presence of ammonium salt. Thepresent inventors further found that, by oxidizing this compound usingan oxidant such as hydrogen peroxide, a nitroxyl radical derivativehaving substituents at position-2 and position-6 can be obtained in ahigh yield. In addition, the present inventors found that, by utilizingammonium salt in which a nitrogen nucleus is labeled with ¹⁵N in theabove-described reaction, a compound in which a nitrogen nucleus of aproduct is labeled can be obtained. Thus, the present invention wasachieved.

That is, the present invention provides a method for producing a2,6-substituted-4-piperidone derivative, a method for producing anitroxyl radical derivative using a compound obtained using the method,a compound obtained using the above-described methods, etc., asdescribed below:

[1] A method for producing a 2,6-substituted-4-piperidone derivativerepresented by the following formula:

[wherein in the formula: R¹, R², R³ and R⁴ are each independently ahydrogen atom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenyl group, a C₂-C₂₀alkynyl group, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ aryl group, a C₆-C₂₀alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀ cycloalkyl group, aC₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl) C₁-C₁₀ alkyl group,and the aforementioned groups may be interrupted by an oxygen atom, anitrogen atom or a sulfur atom; any one of R¹, R², R³ and R⁴ is a groupother than a hydrogen atom; and R¹ and R² and/or R³ and R⁴ may beindependently crosslinked to each other to form a substituted orunsubstituted C₄-C₄₀ monocyclic or polycyclic saturated ring, and thesaturated ring may be interrupted by an oxygen atom, a nitrogen atom ora sulfur atom],wherein the method comprises the step of reacting a triacetoneaminederivative represented by the following formula:

[wherein in the formula: R is a hydrogen atom or a C₁-C₆ alkyl group;and R′s are each independently a C₁-C₆ alkyl group],(in which the case where the triacetoneamine derivative represented bythe formula (1) is completely identical to the2,6-substituted-4-piperidone derivative represented by the formula (1)is excluded), with a ketone or aldehyde derivative represented by thefollowing formula:

[wherein in the formula: R⁵ and R⁶ are each independently a hydrogenatom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenyl group, a C₂-C₂₀ alkynylgroup, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ aryl group, a C₆-C₂₀alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀ cycloalkyl group, aC₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl) C₁-C₁₀ alkyl group,and the aforementioned groups may be interrupted by an oxygen atom, anitrogen atom or a sulfur atom; any one of R⁵ and R⁶ is a group otherthan a hydrogen atom; and R⁵ and R⁶ may be crosslinked to each other toform a substituted or unsubstituted C₄-C₄₀ monocyclic or polycyclicsaturated ring, and the saturated ring may be interrupted by an oxygenatom, a nitrogen atom or a sulfur atom], in the presence of ammoniumsalt.[2] The production method according to item [1], wherein in the formula(1), R is a methyl group.[3] The production method according to item [1] or [2], wherein the2,6-substituted-4-piperidone represented by the formula (1) and theammonium salt are ¹⁵N-labeled compounds.[4] A 2,6-substituted-4-piperidone derivative, which is obtained usingthe method according to any one of items [1] to [3].[5] A 2,6-substituted-4-piperidone derivative represented by thefollowing formula:

[wherein in the formula: R^(1′), R^(2′), R^(3′) and R^(4′) are eachindependently a hydrogen atom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenylgroup, a C₂-C₂₀ alkynyl group, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ arylgroup, a C₆-C₂₀ alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀cycloalkyl group, a C₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl)C₁-C₁₀ alkyl group, and the aforementioned groups may be interrupted byan oxygen atom, a nitrogen atom or a sulfur atom; any one of R^(1′),R^(2′), R^(3′) and R^(4′) is a group other than a hydrogen atom; andR^(1′) and R^(2′) and/or R^(3′) and R^(4′) are independently crosslinkedto each other to form a substituted or unsubstituted C₄-C₄₀ monocyclicor polycyclic saturated ring, and the saturated ring may be interruptedby an oxygen atom, a nitrogen atom or a sulfur atom (wherein the casewhere R^(1′) and R^(2′) and/or R^(3′) and R^(4′) form a cyclohexane ringwith no substituent is excluded)].[6] The 2,6-substituted-4-piperidone derivative according to item [5],which is represented by the following formula:

[wherein in the formula: X¹ and X² are each independently a substitutedor unsubstituted C₄-C₂₀ cycloalkylene group which may be interrupted byan oxygen atom, a nitrogen atom or a sulfur atom; carbonyl group;acetamide group; sulfonyl group; sulfinyl group; an oxygen atom; or asulfur atom].[7] A 2,6-substituted-4-piperidone derivative represented by thefollowing formula:

[8] The 2,6-substituted-4-piperidone derivative according to any one ofitems [4] to [7], wherein a nitrogen nucleus is labeled with ¹⁵N.[9] A method for producing a nitroxyl radical derivative represented bythe following formula:

[wherein in the formula: R¹, R², R³ and R⁴ are each independently ahydrogen atom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenyl group, a C₂-C₂₀alkynyl group, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ aryl group, a C₆-C₂₀alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀ cycloalkyl group, aC₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl) C₁-C₁₀ alkyl group,and the aforementioned groups may be interrupted by an oxygen atom, anitrogen atom or a sulfur atom; any one of R¹, R², R³ and R⁴ is a groupother than a hydrogen atom; and R¹ and R² and/or R³ and R⁴ may beindependently crosslinked to each other to form a substituted orunsubstituted C₄-C₄₀ monocyclic or polycyclic saturated ring, and thesaturated ring may be interrupted by an oxygen atom, a nitrogen atom ora sulfur atom],wherein the method comprises the step of producing a nitroxyl radical byoxidizing an amino group of a 2,6-substituted-4-piperidone derivativeobtained using the method according to any one of items [1] to [3],which is represented by the following formula:

[wherein in the formula: R¹, R², R³ and R⁴ mean the same as describedabove].[10] The production method according to item [9], wherein the nitroxylradical derivative represented by the formula (II), the ammonium saltand the 2,6-substituted-4-piperidone derivative represented by theformula (1) are ¹⁵N-labeled compounds.[11] A nitroxyl radical derivative, which is obtained using the methodaccording to item [9] or [10].[12] A nitroxyl radical derivative represented by the following formula:

[wherein in the formula: R^(1′), R^(2′), R^(3′) and R^(4′) are eachindependently a hydrogen atom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenylgroup, a C₂-C₂₀ alkynyl group, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ arylgroup, a C₆-C₂₀ alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀cycloalkyl group, a C₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl)C₁-C₁₀ alkyl group, and the aforementioned groups may be interrupted byan oxygen atom, a nitrogen atom or a sulfur atom; any one of R^(1′),R^(2′), R^(3′) and R^(4′) is a group other than a hydrogen atom; andR^(1′) and R^(2′) and/or R^(3′) and R^(4′) are independently crosslinkedto each other to form a substituted or unsubstituted C₄-C₄₀ monocyclicor polycyclic saturated ring, and the saturated ring may be interruptedby an oxygen atom, a nitrogen atom or a sulfur atom (wherein the casewhere R^(1′) and R^(2′) and/or R^(3′) and R^(4′) form a cyclohexane ringwith no substituent is excluded)].[13] The nitroxyl radical derivative according to item [12], which isrepresented by the following formula:

[wherein in the formula: X¹ and X² are each independently a substitutedor unsubstituted C₄-C₂₀ cycloalkylene group which may be interrupted byan oxygen atom, a nitrogen atom or a sulfur atom; carbonyl group;acetamide group; sulfonyl group; sulfinyl group; an oxygen atom; or asulfur atom].[14] A nitroxyl radical derivative represented by the following formula:

[15] The nitroxyl radical derivative according to any one of items [11]to [14], wherein a nitrogen nucleus is labeled with ¹⁵N.

According to the present invention, a 2,6-substituted-4-piperidonederivative can be obtained using a simple method. Further, according toa preferred embodiment of the present invention, by oxidizing an aminogroup of the obtained 2,6-substituted-4-piperidone derivative, anitroxyl radical derivative, in which a TEMPO-based compound hassubstituents at position-2 and position-6, can be obtained in a highyield. According to the method, by using ammonium salt in which anitrogen nucleus is labeled with ¹⁵N as a raw material, a labeledcompound of interest can be easily obtained. The obtained nitroxylradical derivative and a labeled compound thereof are particularlyuseful, for example, as a contrast agent for following a free radicalreaction in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph for comparison between reactivities of 3 types ofTEMPO-based nitroxyl radical derivatives, in which substituents atposition-2 and position-6 in one derivative are different from those inanother derivative, to hydroxyl radical.

FIG. 2 is a graph for comparison between reactivities of 3 types ofTEMPO-based nitroxyl radical derivatives, in which substituents atposition-2 and position-6 in one derivative are different from those inanother derivative, to ascorbic acid.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail. First,the method for producing a 2,6-substituted-4-piperidone derivative ofthe present invention will be described.

A. Method for producing a 2,6-substituted-4-piperidone derivative

The method for producing a 2,6-substituted-4-piperidone derivative ofthe present invention is a method for producing a2,6-substituted-4-piperidone derivative represented by the followingformula:

[wherein in the formula: R¹, R², R³ and R⁴ are each independently ahydrogen atom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenyl group, a C₂-C₂₀alkynyl group, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ aryl group, a C₆-C₂₀alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀ cycloalkyl group, aC₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl) C₁-C₁₀ alkyl group,and the aforementioned groups may be interrupted by an oxygen atom, anitrogen atom or a sulfur atom; any one of R¹, R², R³ and R⁴ is a groupother than a hydrogen atom; and R¹ and R² and/or R³ and R⁴ may beindependently crosslinked to each other to form a substituted orunsubstituted C₄-C₄₀ monocyclic or polycyclic saturated ring, and thesaturated ring may be interrupted by an oxygen atom, a nitrogen atom ora sulfur atom],wherein the method comprises the step of reacting a triacetoneaminederivative represented by the following formula:

[wherein in the formula: R is a hydrogen atom or a C₁-C₆ alkyl group;and R′s are each independently a C₁-C₆ alkyl group],(in which the case where the triacetoneamine derivative represented bythe formula (1) is completely identical to the2,6-substituted-4-piperidone derivative represented by the formula (1)is excluded), with a ketone or aldehyde derivative represented by thefollowing formula:

[wherein in the formula: R⁵ and R⁶ are each independently a hydrogenatom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenyl group, a C₂-C₂₀ alkynylgroup, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ aryl group, a C₆-C₂₀alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀ cycloalkyl group, aC₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl) C₁-C₁₀ alkyl group,and the aforementioned groups may be interrupted by an oxygen atom, anitrogen atom or a sulfur atom; any one of R⁵ and R⁶ is a group otherthan a hydrogen atom; and R⁵ and R⁶ may be crosslinked to each other toform a substituted or unsubstituted C₄-C₄₀ monocyclic or polycyclicsaturated ring, and the saturated ring may be interrupted by an oxygenatom, a nitrogen atom or a sulfur atom], in the presence of ammoniumsalt (hereinafter also referred to as “step A”).

In the step A, as shown in the reaction scheme below, a triacetoneaminederivative (a compound represented by formula (1)) is reacted with aketone or aldehyde derivative represented by formula (2) in the presenceof ammonium salt (a compound represented by formula (1)), therebyobtaining a 2,6-substituted-4-piperidone derivative represented byformula (1):

[wherein in the formulae: R¹, R², R³, R⁴, R⁵, R⁶, R and R′ mean the sameas described above; and Y is a halogen atom, an acetyloxy group, atrifluoroacetyloxy group, formate (HCO₂) or hydrogensulfate (HSO₄)].

The mechanism of the above-described reaction is not clear. However,when the reaction is performed using ammonium salt labeled with ¹⁵N,replacement of a nitrogen atom of a substrate occurs and a compoundrepresented by formula (1) in which a nitrogen nucleus is labeled with¹⁵N is obtained in a high yield. Therefore, it is inferred that areaction with a ketone or aldehyde derivative proceeds accompanied bytransfer of an ammonia compound, as shown below.

As described above, according to the present invention, by using thesimple method in which a triacetoneamine derivative is reacted with aketone or aldehyde derivative represented by formula (2) in the presenceof ammonium salt, a substituent can be introduced into both or at leastone of position-2 and position-6, and in addition, according to need, anitrogen nucleus of a substrate can be labeled with ¹⁵N. The2,6-substituted-4-piperidone derivative thus obtained is useful as anintermediate compound of a nitroxyl radical derivative or a labeledcompound thereof, since such a nitroxyl radical derivative or a labeledcompound thereof of interest can be easily obtained by oxidizing the2,6-substituted-4-piperidone derivative according to the ordinarymethod.

(Compounds as Raw Materials)

Hereinafter, compounds as raw materials to be used in the step A will bedescribed.

The triacetoneamine derivative to be used in the present invention is acompound represented by the following formula:

[wherein in the formula: R is a hydrogen atom or a C₁-C₆ alkyl group;and R′s are each independently a C₁-C₆ alkyl group] (in which the casewhere the compound is completely identical to the2,6-substituted-4-piperidone derivative represented by theaforementioned formula (1) is excluded). The “case where completelyidentical” means the case where not only the type of substituent and thebinding position, but also the steric structure, the presence or absenceof isotope, etc. of the two compounds are the same.

In this regard, examples of C₁-C₆ alkyl groups include methyl group,ethyl group, propyl group, isopropyl group, n-butyl group, s-butylgroup, t-butyl group, pentyl group and hexyl group. Among them, R ispreferably a hydrogen atom, a methyl group or an ethyl group, andparticularly preferably a methyl group. R′ is preferably a methyl groupor an ethyl group, and particularly preferably a methyl group.

Among triacetoneamine derivatives represented by the above-describedformula (1), triacetoneamine (a compound in which R is a hydrogen atomand all R′s are methyl groups) or N-methyl-triacetoneamine (a compoundin which R is a methyl group and all R′s are methyl groups) ispreferably used on the point that these compounds are commerciallyavailable and can be easily obtained. In particular, since the2,6-substituted-4-piperidone derivative represented by formula (1) canbe obtained in a higher yield, R is preferably a methyl group, andN-methyl-triacetoneamine is preferably used. These compounds are offeredcommercially, for example, by Aldrich.

Regarding the ketone or aldehyde derivative represented by theabove-described formula (2), in the formula: R⁵ and R⁶ are eachindependently a hydrogen atom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenylgroup, a C₂-C₂₀ alkynyl group, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ arylgroup, a C₆-C₂₀ alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀cycloalkyl group, a C₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl)C₁-C₁₀ alkyl group, and the aforementioned groups may be interrupted byan oxygen atom, a nitrogen atom or a sulfur atom; any one of R⁵ and R⁶is a group other than a hydrogen atom; and R⁵ and R⁶ may be crosslinkedto each other to form a substituted or unsubstituted C₄-C₄₀ monocyclicor polycyclic saturated ring, and the saturated ring may be interruptedby an oxygen atom, a nitrogen atom or a sulfur atom. In particular, itis preferred that R⁵ and R⁶ are crosslinked to form a 5 to 8-memberedring.

In the present invention, the ketone or aldehyde derivatives representedby formula (2) may be used solely or in combination.

In this specification, the C₁-C₂₀ alkyl group is preferably a C₁-C₁₀alkyl group. Examples of alkyl groups include methyl group, ethyl group,propyl group, isopropyl group, n-butyl group, s-butyl group, t-butylgroup, pentyl group, hexyl group, octyl group, nonyl group, decyl group,undecyl group and dodecyl group.

The C₂-C₂₀ alkenyl group is preferably a C₂-C₁₀ alkenyl group. Examplesof alkenyl groups include vinyl group, allyl group, propenyl group,isopropenyl group, 2-methyl-1-propenyl group, 2-methylallyl group and2-butenyl group.

The C₂-C₂₀ alkynyl group is preferably a C₂-C₁₀ alkynyl group. Examplesof alkynyl groups include ethynyl group, propynyl group and butynylgroup.

The C₄-C₂₀ alkyldienyl group is preferably a C₄-C₁₀ alkyldienyl group.Examples of alkyldienyl groups include 1,3-butadienyl group.

The C₆-C₁₈ aryl group is preferably a C₆-C₁₀ aryl group. Examples ofaryl groups include phenyl group, 1-naphthyl group, 2-naphthyl group,indenyl group, biphenyl group, anthryl group and phenanthryl group.

The C₆-C₂₀ alkylaryl group is preferably a C₆-C₁₀ alkylaryl group.Examples of alkylaryl groups include o-tolyl group, m-tolyl group,p-tolyl group, 2,3-xylyl group, 2,5-xylyl group, o-cumenyl group,m-cumenyl group, p-cumenyl group and mesityl group.

The C₆-C₂₀ arylalkyl group is preferably a C₆-C₁₀ arylalkyl group.Examples of arylalkyl groups include benzyl group, phenethyl group,1-naphthylmethyl group, 2-naphthylmethyl group, 1-phenylethyl group,phenylpropyl group, phenylbutyl group, phenylpentyl group, phenylhexylgroup, methylbenzyl group, dimethylbenzyl group, trimethylbenzyl group,ethylbenzyl group, methylphenethyl group, dimethylphenethyl group anddiethylbenzyl group.

C₄-C₂₀ cycloalkyl group is preferably a C₄-C₁₀ cycloalkyl group.Examples of cycloalkyl groups include cyclopropyl group, cyclobutylgroup, cyclopentyl group, cyclohexyl group, cycloheptyl group, andcyclooctyl group.

C₄-C₂₀ cycloalkenyl group is preferably a C₄-C₁₀ cycloalkenyl group.Examples of cycloalkenyl groups include cyclopropenyl group,cyclobutenyl group, cyclopentenyl group, cyclopentadienyl group andcyclohexenyl group.

The (C₃-C₁₀ cycloalkyl) C₁-C₁₀ alkyl group is a C₁-C₁₀ alkyl grouphaving a C₃-C₁₀ cycloalkyl group. Examples thereof include groupsconsisting of a combination of a cycloalkyl group and an alkyl groupselected from the aforementioned cycloalkyl groups and alkyl groups.

Note that the above-described groups may be interrupted by an oxygenatom, a nitrogen atom or a sulfur atom. When the above-described groupsare interrupted by a nitrogen atom, they are preferably a grouprepresented by —N(Z)— (wherein in the formula, Z is a hydrogen atom or aC₁-C₂₀ hydrocarbon group). In this regard, preferred examples ofhydrocarbon groups include alkyl group, alkenyl group and alkynyl group.When the above-described groups are interrupted by a sulfur atom, theyare preferably a group represented by —S(O)n- (wherein in the formula, nis 0, 1 or 2).

As the ketone or aldehyde derivative represented by formula (2), forexample, compounds as shown below can be used, and they are commerciallyavailable and can be easily obtained.

Further, R⁵ and R⁶ may be crosslinked to each other to form asubstituted or unsubstituted C₄-C₄₀ monocyclic or polycyclic saturatedring, and the saturated ring may be interrupted by an oxygen atom, anitrogen atom or a sulfur atom. The aforementioned monocyclic orpolycyclic saturated ring is preferably a C₄-C₂₀ monocyclic orpolycyclic saturated ring. In particular, it is preferred that R⁵ and R⁶are crosslinked to each other to form a 5 to 8-membered ring.

Regarding the 2,6-substituted-4-piperidone derivative obtained in thestep A, R¹ and R² and/or R³ and R⁴ in formula (1) may be independentlycrosslinked to each other to form a substituted or unsubstituted C₄-C₄₀monocyclic or polycyclic saturated ring, as in the case of R⁵ and R⁶,and the saturated ring may be interrupted by an oxygen atom, a nitrogenatom or a sulfur atom. The aforementioned monocyclic or polycyclicsaturated ring is preferably a C₄-C₂₀ monocyclic or polycyclic saturatedring, and it is more preferred that they are crosslinked to form a 5 to8-membered ring.

In this regard, examples of substituents include C₁-C₁₀ alkyl group,C₂-C₁₀ alkenyl group, C₂-C₁₀ alkynyl group, C₄-C₁₀ alkyldienyl group,C₆-C₁₀ aryl group, C₆-C₁₀ alkylaryl group, C₆-C₁₀ arylalkyl group,C₄-C₁₀ cycloalkyl group, C₄-C₁₀ cycloalkenyl group, (C₃-C₁₀ cycloalkyl)C₁-C₁₀ alkyl group, carbonyl group, amino group and halogen atom.

Examples of such ketone derivatives include a compound represented bythe following formula:

[wherein in the formula, X is a substituted or unsubstituted alkylenegroup, carbonyl group, acetamide group, sulfonyl group or sulfinylgroup, or an oxygen atom, or a sulfur atom].

Examples of substituents of the alkylene group include C₁-C₁₀ alkylgroup, C₂-C₁₀ alkenyl group, C₂-C₁₀ alkynyl group, C₄-C₁₀ alkyldienylgroup, C₆-C₁₀ aryl group, C₆-C₁₀ alkylaryl group, C₆-C₁₀ arylalkylgroup, C₄-C₁₀ cycloalkyl group, C₄-C₁₀ cycloalkenyl group and (C₃-C₁₀cycloalkyl) C₁-C₁₀ alkyl group. The alkylene group is preferably aC₁-C₂₀ alkylene group, and more preferably a C₁-C₁₀ alkylene group.Specifically, methylene group, ethylene group, trimethylene group,propylene group, etc. are preferred. Among them, methylene group isparticularly preferred.

When X is a substituted or unsubstituted alkylene group, X will have twosubstituents. These substituents may be crosslinked to each other toform a substituted or unsubstituted C₄-C₂₀ monocyclic or polycyclicsaturated ring, and the saturated ring may be interrupted by an oxygenatom, a nitrogen atom or a sulfur atom. As the substituents, the samesubstituents as those for R⁵ and R⁶ can be used. When the aforementionedsaturated ring is interrupted by a nitrogen atom, it is preferably agroup represented by —N(Z)— (wherein in the formula, Z is a hydrogenatom or a C₁-C₂₀ hydrocarbon group). In this regard, preferred examplesof hydrocarbon groups include alkyl group, alkenyl group and alkynylgroup. When these groups are interrupted by a sulfur atom, they arepreferably a group represented by —S(O)n- (wherein in the formula, n is0, 1 or 2).

As the compound represented by formula (ii), for example, compounds asshown below can be used, and they are commercially available and can beeasily obtained.

The use amount of the ketone or aldehyde derivative represented byformula (2) is preferably 1.0 to 10.0 equivalents, more preferably 2.0to 5.0 equivalents, and particularly preferably 2.0 to 3.0 equivalentsper 1 equivalent of triacetoneamine derivative.

Ammonium salt to be used in the present invention is not particularlylimited, but those which can develop a reaction under relatively mildconditions are preferably used. For example, compounds represented bythe following formula:

NH₄Y  (i)

[wherein in the formula, Y is a halogen atom, an acetyloxy group, atrifluoroacetyloxy group, formate (HCO₂) or hydrogensulfate (HSO₄)] canbe used. In this regard, examples of halogen atoms include fluorine,chlorine, bromine and iodine, and chlorine or bromine is particularlypreferred. More specifically, ammonium chloride salt, ammonium bromidesalt, ammonium acetate salt, ammonium trifluoroacetate salt, ammoniumformate salt, ammonium hydrogen sulfate salt, etc. are preferably used.

When obtaining a labeled compound, ammonium salt in which a nitrogennucleus is labeled with ¹⁵N is used. By using a ¹⁵N-labeled compound ofammonium salt, the 2,6-substituted-4-piperidone derivative can be easilylabeled.

The use amount of ammonium salt is preferably 2.0 to 10.0 equivalents,more preferably 3.0 to 8.0 equivalents, and particularly preferably 5.0to 7.0 equivalents per 1 equivalent of triacetoneamine derivative.

In the step A, compounds as raw materials such as the triacetoneaminederivative, the ketone or aldehyde derivative represented by formula (2)and ammonium salt are mixed in a solvent to cause a reaction. A reactionsolvent is not particularly limited as long as it is inactive withrespect to these compounds. For example, organic solvents such as DMSO,DMF, THF, dioxane, methanol, ethanol and propanol are preferably used.

(Reaction Conditions)

Next, reaction conditions will be described.

The reaction in the step A can be performed under relatively mildconditions. The reaction temperature is preferably room temperature to90° C., more preferably 50 to 80° C., and particularly preferably 60 to70° C.

The reaction time may be suitably determined based on confirmation ofextent of reaction, but is usually about 3 to 20 hours, preferably 5 to15 hours, and more preferably 6 to 10 hours.

Further, the step A can be carried out under increased pressure, reducedpressure or atmospheric pressure. However, in view of easiness ofoperation, it is desired that the step A is carried out underatmospheric pressure.

After completion of the reaction, a product is separated/extracted fromthe reaction solution and purified according to the ordinary method,thereby obtaining a 2,6-substituted-4-piperidone derivative of interest.

In the step A, using the simple method as described above, substituentscan be introduced into position-2 and position-6 of 4-piperidone.Therefore, it is possible to easily obtain 2,6-substituted-4-piperidonederivatives with various properties. Further, by using ammonium salt inwhich a nitrogen nucleus is labeled with ¹⁵N, a ¹⁵N-labeled compound of2,6-substituted-4-piperidone derivative can be obtained in a high yield.Therefore, the present invention has the advantage that the productionprocess can be more simplified compared to the conventional method.Moreover, the ¹⁵N-labeled compound of 2,6-substituted-4-piperidonederivative can also be easily produced in a high yield by changing oneof the compounds as raw materials to be used in the step A to a¹⁵N-labeled compound thereof. The present invention also includescompounds obtained in such a manner.

In particular, in the present invention, 2,6-substituted-4-piperidonederivatives represented by the following formula:

[wherein in the formula: R^(1′), R^(2′), R^(3′) and R^(4′) are eachindependently a hydrogen atom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenylgroup, a C₂-C₂₀ alkynyl group, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ arylgroup, a C₆-C₂₀ alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀cycloalkyl group, a C₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl)C₁-C₁₀ alkyl group, and the aforementioned groups may be interrupted byan oxygen atom, a nitrogen atom or a sulfur atom; any one of R^(1′),R^(2′), R^(3′) and R^(4′) is a group other than a hydrogen atom; andR^(1′) and R^(2′) and/or R^(3′) and R^(4′) are independently crosslinkedto each other to form a substituted or unsubstituted C₄-C₄₀ monocyclicor polycyclic saturated ring, and the saturated ring may be interruptedby an oxygen atom, a nitrogen atom or a sulfur atom (wherein the casewhere R^(1′) and R^(2′) and/or R^(3′) and R^(4′) form a cyclohexane ringwith no substituent is excluded)] are preferred. There is a possibilitythat these compounds exhibit properties different from those ofconventional compounds, and therefore, application to various uses isexpected.

Among such compounds, compounds represented by the following formula:

[wherein in the formula, X¹ and X² are each independently a substitutedor unsubstituted C₄-C₂₀ cycloalkylene group which may be interrupted byan oxygen atom, a nitrogen atom or a sulfur atom; carbonyl group;acetamide group; sulfonyl group; sulfinyl group; an oxygen atom; or asulfur atom] are preferred. Note that X¹ and X² are the limitation ofthe aforementioned X, and examples of substituents for X¹ and X² andpreferred examples of X¹ and X² are the same as those of theaforementioned X. Therefore, explanation thereof is omitted herein.

More specifically, preferred examples of 2,6-substituted-4-piperidonederivatives obtained using the method of the present invention includethe following compounds:

Among the above-described 2,6-substituted-4-piperidone derivatives,¹⁵N-labeled compounds thereof in which a nitrogen nucleus is labeledwith ¹⁵N are preferred.

B. Method for Producing Nitroxyl Radical Derivative

Next, the method for producing a nitroxyl radical derivative of thepresent invention will be described.

The present invention provides a method for producing a nitroxyl radicalderivative represented by the following formula:

[wherein in the formula: R¹, R², R³ and R⁴ are each independently ahydrogen atom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenyl group, a C₂-C₂₀alkynyl group, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ aryl group, a C₆-C₂₀alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀ cycloalkyl group, aC₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl) C₁-C₁₀ alkyl group,and the aforementioned groups may be interrupted by an oxygen atom, anitrogen atom or a sulfur atom; any one of R¹, R², R³ and R⁴ is a groupother than a hydrogen atom; and R¹ and R² and/or R³ and R⁴ may beindependently crosslinked to each other to form a substituted orunsubstituted C₄-C₄₀ monocyclic or polycyclic saturated ring, and thesaturated ring may be interrupted by an oxygen atom, a nitrogen atom ora sulfur atom],wherein the method comprises the step of oxidizing an amino group of the2,6-substituted-4-piperidone derivative obtained in the aforementionedstep A, which is represented by the following formula:

[wherein in the formula, R¹, R², R³ and R⁴ mean the same as describedabove], to produce a nitroxyl radical (hereinafter also referred to as“step B”).

In the step B, as shown in the reaction scheme below, an amino group ofthe 2,6-substituted-4-piperidone derivative obtained in theaforementioned step A of the “method for producing a2,6-substituted-4-piperidone derivative” is oxidized, thereby obtaininga nitroxyl radical:

[wherein in the formula, R¹, R², R³ and R⁴ mean the same as describedabove].

According to the step B, substituents can be introduced into position-2and position-6 of a TEMPO-based compound using a simple method. Further,when using a labeled compound of the 2,6-substituted-4-piperidonederivative, a labeled compound of a nitroxyl radical derivative ofinterest can be obtained in a high yield.

(Compounds as Raw Materials)

Examples of oxidants to be used in the step B include hydrogen peroxide,perbenzoic acids such as m-chloroperbenzoic acid, peracetic acid,periodic acid and oxonic acid. Among them, hydrogen peroxide ispreferably used, and 30% hydrogen peroxide is particularly preferablyused. The use amount of the oxidant is preferably 3 to 50 equivalents,more preferably 5 to 15 equivalents, and particularly preferably 7 to 10equivalents per 1 equivalent of 2,6-substituted-4-piperidone.

In the step B, an oxidation catalyst can be employed for a combinationuse. Examples of oxidation catalysts that can be used in the step Binclude sodium tungstate and methyltrioctylammonium hydrogen sulfate.

The reaction is preferably performed in a solvent. The solvent is notparticularly limited as long as it is inactive with respect to the2,6-substituted-4-piperidone derivative. Preferred examples thereofinclude organic solvent such as alcohols (for example, alcohol andmethanol), chloroform and dichloromethane.

(Reaction Conditions)

Next, reaction conditions in the step B will be described.

In the step B, the reaction temperature is preferably 0 to 30° C., morepreferably 15 to 30° C., and particularly preferably 20 to 25° C.

The reaction time may be suitably determined based on confirmation ofextent of reaction, but is usually about 10 to 40 hours, preferably 15to 30 hours, and more preferably 20 to 25 hours.

Further, the step B can be carried out under increased pressure, reducedpressure or atmospheric pressure. However, in view of easiness ofoperation, it is desired that the step B is carried out underatmospheric pressure.

After completion of the reaction, a product is separated/extracted fromthe reaction solution and purified according to the ordinary method,thereby obtaining a nitroxyl radical derivative of interest.

According to the method of the present invention, by using the simplemethod in which an amino group of the 2,6-substituted-4-piperidonederivative is oxidized, the nitroxyl radical derivative represented bythe following formula:

[wherein in the formula: R¹, R², R³ and R⁴ mean the same as describedabove] can be obtained in a high yield. Moreover, by using a ¹⁵N-labeledcompound of the 2,6-substituted-4-piperidone derivative, a ¹⁵N-labeledcompound of the nitroxyl radical derivative can also be easily obtainedin a high yield. The present invention also includes compounds obtainedin such a manner.

In particular, in the present invention, nitroxyl radical derivativesrepresented by the following formula:

[wherein in the formula: R^(1′), R^(2′), R^(3′) and R^(4′) are eachindependently a hydrogen atom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenylgroup, a C₂-C₂₀ alkynyl group, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ arylgroup, a C₆-C₂₀ alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀cycloalkyl group, a C₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl)C₁-C₁₀ alkyl group, and the aforementioned groups may be interrupted byan oxygen atom, a nitrogen atom or a sulfur atom; any one of R^(1′),R^(2′), R^(3′) and R^(4′) is a group other than a hydrogen atom; andR^(1′) and R^(2′) and/or R^(3′) and R^(4′) are independently crosslinkedto each other to form a substituted or unsubstituted C₄-C₄₀ monocyclicor polycyclic saturated ring, and the saturated ring may be interruptedby an oxygen atom, a nitrogen atom or a sulfur atom (wherein the casewhere R^(1′) and R^(2′) and/or R^(3′) and R^(4′) independently than acyclohexane ring with no substituent is excluded)] are preferred. Thereis a possibility that these compounds exhibit properties different fromthose of conventional compounds, and therefore, application to varioususes is expected.

Among such compounds, nitroxyl radical derivatives represented by thefollowing formula:

[wherein in the formula, X¹ and X² are each independently a substitutedor unsubstituted C₄-C₂₀ cycloalkylene group which may be interrupted byan oxygen atom, a nitrogen atom or a sulfur atom; carbonyl group;acetamide group; sulfonyl group; sulfinyl group; an oxygen atom; or asulfur atom] are preferred. Note that X¹ and X² are the limitation ofthe aforementioned X, and examples of substituents for X¹ and X² andpreferred examples of X¹ and X² are the same as those of theaforementioned X. Therefore, explanation thereof is omitted herein.

More specifically, preferred examples of nitroxyl radical derivativesobtained using the method of the present invention include the followingcompounds:

Among the above-described nitroxyl radical derivatives, ¹⁵N-labeledcompounds thereof in which a nitrogen nucleus is labeled with ¹⁵N arepreferred.

The nitroxyl radical derivative of the present invention can be widelyused as an antioxidative agent, a chemical cell, a polymerization agentor the like. In particular, the nitroxyl radical derivative of thepresent invention is useful as a contrast agent for following a freeradical reaction in vivo, utilizing its high sensitivity to a freeradical such as active oxygen. Regarding a nitroxyl radical derivativeadministered to a living body, the distribution, etc. therein varydepending on the basic structure and the type of substituent. Whenattention is focused on this point, it is possible to perform imageanalysis in vivo by labeling a nitrogen nucleus of each of nitroxylradical derivatives having different distribution properties in vivowith ¹⁴N or ¹⁵N to distinguish the information obtained by ¹⁴N from theinformation obtained by ¹⁵N for analysis. The present inventors alreadydeveloped the simultaneous separating imaging method using a ¹⁴N-labeledor ¹⁵N-labeled compound of the nitroxyl radical derivative (seeNon-patent document 1: H. Utsumi, K. Yamada, K. Ichikawa, K. Sakai, Y.Kinoshita, S. Matsumoto and M. Nagai, PNAS, 103, 1463 (2006)). Accordingto the present invention, nitroxyl radical derivatives with variousdistribution properties in vivo, which are useful for the image analysisin vivo, can be produced using the simple method.

EXAMPLES

Hereinafter, the present invention will be more specifically describedby way of examples. However, the present invention is not limited onlyto these examples.

Example 1 Synthesis of 2,6-spirocyclohexyl piperidine-4-one 1-oxyl (StepA)

1.55 g (1 eq.) of triacetoneamine, 3.21 g (6 eq.) of NH₄Cl and 2.94 g (3eq.) of cyclohexanone were mixed in a DMSO solvent (20 ml), and themixture was heated and stirred at 60° C. for 15 to 20 hours. After areaction, the solution was diluted with water (40 ml), and was madeacidic with 7% hydrochloric acid (10 ml). The neutral portion wasextracted and removed using ether. The pH of the mother liquid wasadjusted to 9 to 10 using 10% aqueous solution of potassium carbonate,and extraction with ethyl acetate was carried out 3 times. The extractedethyl acetate layers were gathered, washed with saline twice, andthereafter dried with anhydrous sodium sulfate. The solvent was removedunder reduced pressure. The obtained oily substance was purified usingsilica gel column chromatography (developing solvent; ethylacetate:hexane). The obtained crystal was recrystallized fromhexane-ethyl acetate, thereby obtaining 380 mg of 2,6-spirocyclohexylpiperidine-4-one (yield: 16%).

Melting point: 103° C.

¹HNMR (CDCl₃) δ 1.35˜1.60 (20H), 2.30 (4H, s, —CH₂COCH₂—).

FAB-MS m/z 236.3 (⁺M+1)

(Step B)

235 mg of 2,6-spirocyclohexyl piperidine-4-one obtained in theaforementioned step was dissolved in methanol (10 ml), and to thismixture, 30% H₂O₂ (2 ml), and subsequently Na₂WO₄ (50 mg) dissolved inwater (1 ml) were added, and the mixture was stirred at room temperaturefor 20 hours. After a reaction, 10% acidic aqueous solution of sodiumsulfite (20 ml) was added to the mixture, followed by stirring for 10minutes. After that, water (20 ml) was added thereto, and extractionwith chloroform was carried out 3 times. After dried with salt cake, thesolvent was distilled away under reduced pressure, and the obtainedcrystal was recrystallized from hexane, thereby obtaining 208 mg of2,6-spirocyclohexyl piperidine-4-one 1-oxyl (yield: 83%).

Melting point: 116.5° C.

FAB-MS m/z 251.3 (⁺M+1)

Example 2 Synthesis of 2,6-spirocyclohexyl piperidine-4-one 1-oxyl (StepA)

1.69 g (1 eq.) of N-methyl-triacetoneamine, 3.21 g (6 eq.) of NH₄Cl and2.94 g (3 eq.) of cyclohexanone were mixed in a DMSO solvent (20 ml),and the mixture was stirred at 60° C. for 5 hours. The reaction solutionwas diluted with water (40 ml), and was made acidic with 7% hydrochloricacid (10 ml). The neutral portion was extracted and removed using ether.The pH of the mother liquid was adjusted to 9 to 10 using 10% aqueoussolution of potassium carbonate, and extraction with ethyl acetate wascarried out 3 times. The extracted ethyl acetate phases were gathered,washed with saline twice, and thereafter dried with anhydrous sodiumsulfate. The solvent was removed under reduced pressure. The obtainedoily substance was purified using silica gel column chromatography(developing solvent; hexane:acetate ester). The obtained crystal wasrecrystallized from ethyl acetate-hexane, thereby obtaining 788 mg of2,6-spirocyclohexyl piperidine-4-one (yield: 34%).

(Step B)

295 mg of 2,6-spirocyclohexyl piperidine-4-one 1-oxyl was obtained in amanner similar to that in Step B of Example 1 (yield: 82%).

By comparing the result in Example 1 with that in Example 2, it wasfound that, when using N-methyl-triacetoneamine in place oftriacetoneamine as a starting material, the yield of 2,6-spirocyclohexylpiperidine-4-one in Step A increased about twice.

Example 3 Synthesis ofbis(tetrahydropyran-4′-spiro)-2,6-piperidine-4-one 1-oxyl (Step A)

1.55 g (1 eq.) of triacetoneamine, 3.21 g (6 eq.) of NH₄Cl and 3.00 g (3eq.) of 4-oxotetrahydropyran were mixed in a DMSO solvent (10 ml), andthe mixture was stirred at 60° C. for 15 to 20 hours. The reactionsolution was diluted with water (40 ml), and was made acidic with 7%hydrochloric acid (10 ml). The neutral portion was extracted and removedusing ether. The pH of the mother liquid was adjusted to 9 to 10 using10% aqueous solution of potassium carbonate, and extraction withchloroform was carried out 3 times. The extracted chloroform phases weregathered, washed with saline twice, and thereafter dried with salt caketo remove the solvent. The obtained oily substance was purified usingsilica gel column chromatography (developing solvent; hexane:acetateester:methanol). The obtained crystal was recrystallized fromhexane-acetate ester, thereby obtaining 340 mg ofbis(tetrahydropyran-4′-spiro)-2,6-piperidine-4-one (yield: 14%).

Melting point: 167° C.

¹HNMR (CDCl₃) δ 1.60˜1.68 (8H, brs, CH₂×4), 2.40 (4H, s, —CH₂COCH₂—),3.56 (4H, m, —CH₂OCH₂—), 3.82 (4H, m, —CH₂OCH₂—).

FAB-MS m/z 240.3 (⁺M+1)

(Step B)

130 mg of bis(tetrahydropyran-4′-spiro)-2,6-piperidine-4-one obtained inthe aforementioned step, 30% H₂O₂ (0.8 ml) and Na₂WO₄ (30 mg) were mixedin a solvent of methanol (5 ml), and the mixture was stirred at roomtemperature for 20 hours. 10% acidic aqueous solution of sodium sulfite(10 ml) was added to the mixture, followed by stirring for 10 minutes.After that, water (10 ml) was added thereto, and extraction withchloroform was carried out (3 times). After dried with salt cake, thesolvent was distilled away under reduced pressure, and the obtainedcrystal was recrystallized from ethyl acetate, thereby obtaining 110 mgof bis(tetrahydropyran-4′-spiro)-2,6-piperidine-4-one 1-oxyl.

Melting point: 145.9° C.

FAB-MS m/z 255.3 (⁺M+1)

Example 4 Synthesis of ¹⁵N-labeled compound of 2,6-spirocyclohexylpiperidine-4-one 1-oxyl

A reaction was performed in a manner similar to that in Example 2,except that ¹⁵NH₄Cl in which the nitrogen nucleus is labeled with ¹⁵Nwas used instead of NH₄Cl. As a result, in Step A, 780 mg of ¹⁵N-labeledcompound of 2,6-spirocyclohexyl piperidine-4-one was obtained (yield:34%).

Melting point: 100.4° C.

¹HNMR (CDCl₃) δ 1.35˜1.60 (20H), 2.30 (4H, s, —CH₂COCH₂—).

FAB-MS m/z 237.3 (⁺M+1)

In Step B, 430 mg of 2,6-spirocyclohexyl piperidine-4-one 1-oxyl wasobtained (yield: 80%).

Melting point: 117.4° C.

FAB-MS m/z 251.3 (⁺M+1)

Example 5 Synthesis of ¹⁵N-labeled compound ofbis(tetrahydropyran-4′-spiro)-2,6-piperidine-4-one 1-oxyl

A reaction was performed in a manner similar to that in Example 3,except that 1.69 g (1 eq.) of N-methyl-triacetoneamine and ¹⁵NH₄Cl inwhich the nitrogen nucleus is labeled with ¹⁵N were used instead oftriacetoneamine and NH₄Cl, respectively. As a result, in Step A, 760 mgof bis(tetrahydropyran-4′-spiro)-2,6-piperidine-4-one was obtained(yield: 32%).

Melting point: 167° C.

¹HNMR (CDCl₃) δ 1.60˜1.68 (8H, brs, CH₂×4), 2.40 (4H, s, —CH₂COCH₂—),3.56 (4H, m, —CH₂OCH₂—), 3.82 (4H, m, —CH₂OCH₂—).

FAB-MS m/z 241.2 (⁺M+1)

In Step B, 105 mg of ¹⁵N-labeled compound ofbis(tetrahydropyran-4′-spiro)-2,6-piperidine-4-one 1-oxyl was obtained(yield: 75%).

Example 6 Synthesis ofbis(tetrahydrothiopyran-4′-spiro)-2,6-piperidine-4-one (Step A)

A reaction was performed in a manner similar to that in Step A ofExample 2, except that 3.48 g (3 eq.) of tetrahydrothiopyran-4-one wasused instead of cyclohexanone. As a result, 813 mg ofbis(tetrahydrothiopyran-4′-spiro)-2,6-piperidine-4-one was obtained(yield: 30%).

Melting point: 155 to 157° C.

¹HNMR (CDCl₃) δ 1.76˜1.90 (8H, m, CH₂×4), 2.29 (4H, s, —CH₂COCH₂—),2.42˜2.50 (4H, m, —CH₂SCH₂—), 2.88˜2.96 (4H, m, —CH₂SCH₂—).

FAB-MS m/z 272.2 (⁺M+1)

Example 7 Synthesis ofbis(tetrahydrosulfinylpyran-4′-spiro)-2,6-piperidine-4-one 1-oxyl andbis(tetrahydrosulfonylpyran-4′-spiro)-2,6-piperidine-4-one 1-oxyl

In a manner similar to that in Step B of Example 1, 250 mg ofbis(tetrahydrothiopyran-4′-spiro)-2,6-piperidine-4-one, 30% H₂O₂ (2 ml)and Na₂WO₄ (50 mg) dissolved in water (1.5 ml) were mixed in methanol (8ml), and the mixture was stirred at room temperature for 20 hours. Aftera reaction, 10% acidic aqueous solution of sodium sulfite (20 ml) wasadded to the mixture, followed by stirring for 10 minutes. After that,water (20 ml) was added thereto, and extraction with chloroform wascarried out 3 times. After dried with salt cake, the solvent wasdistilled away under reduced pressure, thereby obtaining a mixture ofwater-soluble bis(tetrahydrosulfinylpyran-4′-spiro)-2,6-piperidine-4-one1-oxyl and bis(tetrahydrosulfonylpyran-4′-spiro)-2,6-piperidine-4-one1-oxyl.

Example 8 Synthesis ofbis(1′-ethylenedioxycyclohexane-4′-spiro)-2,6-piperidine-4-one

A reaction was performed in a manner similar to that in Step A ofExample 2, except that 4.68 g (3 eq.) of 1,4-cyclohexanedionemonoethyleneacetal was used instead of cyclohexanone. As a result, 1.12g of bis(1′-ethylenedioxycyclohexane-4′-spiro)-2,6-piperidine-4-one wasobtained (yield: 32%).

Melting point: 190.6° C.

¹HNMR (CDCl₃) δ 1.50˜1.91 (16H, brt, CH₂×8), 2.35 (4H, s, —CH₂COCH₂—),3.90 (8H, brs, —OCH₂×4).

FAB-MS m/z 352.4 (⁺M+1)

Example 9 Synthesis ofbis(1′-oxocyclohexane-4′-spiro)-2,6-piperidine-4-one

536 mg of bis(1′-ethylenedioxycyclohexane-4′-spiro)-2,6-piperidine-4-onewas dissolved in acetic acid (7 ml) and water (2 ml), 5 drops ofconcentrated hydrochloric acid was added thereto, and the mixture wasstirred at 60° C. for 5 hours. After a reaction, water (30 ml) was addedthereto, and the pH thereof was adjusted to 8 using 5% potassiumcarbonate solution, followed by extraction with chloroform 4 times.After dried with salt cake, the solvent was distilled away under reducedpressure. The obtained oily substance was purified using silica gelcolumn chromatography (developing solvent; acetate ester:hexane). Theobtained crystal was recrystallized from ethyl acetate-hexane, therebyobtaining 330 mg of bis(1′-oxocyclohexane-4′-spiro)-2,6-piperidine-4-one(yield: 82%).

Melting point: 155.3° C.

¹HNMR (CDCl₃) δ 1.82˜1.94 (4H, m, CH₂×2), 2.00˜2.06 (4H, m, CH₂×2),2.26˜2.36 (4H, m, —CH₂COCH₂—), 2.48 (4H, d, —CH₂COCH₂—), 2.58˜2.68 (4H,m, —CH₂COCH₂—).

FAB-MS m/z 264.2 (⁺M+1)

Example 10 Synthesis ofbis(1′-oxocyclohexane-4′-spiro)-2,6-piperidine-4-one 1-oxyl

In a manner similar to that in Step B of Example 1, using 215 mg ofbis(1′-oxocyclohexane-4′-spiro)-2,6-piperidine-4-one, 170 mg ofbis(1′-oxocyclohexane-4′-spiro)-2,6-piperidine-4-one 1-oxyl was obtained(yield: 75%).

Melting point: 161.5° C.

FAB-MS m/z 279.2 (⁺M+1)

Example 11 Synthesis of 2,6-dimethyl-2,6-benzyloxymethyl-4-piperidoneand 2,2,6-trimethyl-benzyloxymethyl-4-piperidone

A reaction was performed in a manner similar to that in Step A ofExample 2, except that 4.92 g (3 eq.) of benzyloxyacetone was usedinstead of cyclohexanone. As a result,2,6-dimethyl-2,6-benzyloxymethyl-4-piperidone and2,2,6-trimethyl-benzyloxymethyl-4-piperidone were obtained. Oilysubstances were separated using silica gel column chromatography(developing solvent; hexane:ethyl acetate), thereby obtaining 410 mg ofoily substance of 2,6-dimethyl-2,6-benzyloxymethyl-4-piperidone (yield:12%) and 512 mg of oily substance of2,2,6-trimethyl-benzyloxymethyl-4-piperidone (yield: 20%).

2,6-dimethyl-2,6-benzyloxymethyl-4-piperidone

¹HNMR (CDCl₃) δ 1.08˜1.15 (m) 6H (CH₃×2), 2.10˜2.50 (4H, m, —CH₂COCH₂—),3.05˜3.50 (4H, m, —OCH₂—×2), 4.20˜4.60 (4H, m, CH₂O×2), 7.30 (10H, brs,aromatic-H).

FAB-MS m/z 368.3 (⁺M+1)

2,2,6-trimethyl-benzyloxymethyl-4-piperidone

¹HNMR (CDCl₃) δ 1.05˜1.13 (9H, m, CH₃×3), 2.08˜2.30 (4H, m, —CH₂COCH₂—),2.60 (1H, m, N—CH), 3.18˜3.30 (2H, m, —CH₂O—), 4.40˜4.60 (2H, m,—CH₂O—), 7.30 (5H, brs, aromatic H).

FAB-MS m/z 262.3 (⁺M+1)

Example 12 Synthesis of 2,6-di-t-butyl-4-piperidone and2,2-dimethyl-6-t-butyl-4-piperidone

A reaction was performed in a manner similar to that in Step A ofExample 2, except that 2.58 g (3 eq.) of trimethyacetaldehyde was usedinstead of cyclohexanone. As a result, 2,6-di-t-butyl-4-piperidone and2,2-dimethyl-6-t-butyl-4-piperidone were obtained. Oily substance wasseparated using silica gel column chromatography (developing solvent;hexane:ethyl acetate), thereby obtaining 260 mg of2,6-di-t-butyl-4-piperidone (crystal obtained by recrystallization fromhexane; yield: 10%; melting point: 55.4° C.) and 300 mg of oilysubstance of 2,2-dimethyl-6-t-butyl-4-piperidone (yield: 12%).

2,6-di-t-butyl-4-piperidone

¹HNMR (CDCl₃) δ 0.90 (18H, s, CH₃×6), 2.05 (2H, brt, J=3), 2.35 (2H,brd, J=3), 2.42 (2H, brd, J=3).

FAB-MS m/z 212.3 (⁺M+1)

2,2-dimethyl-6-t-butyl-4-piperidone

¹HNMR (CDCl₃) δ 0.90 (9H, s, CH₃×3), 1.05 (3H, s, CH₃), 1.10 (3H, s,CH₃), 2.00 (1H, t, J=3.0), 2.20 (2H, brs), 2.35 (1H, d, J=3.0), 2.85(1H, d, J=3.0, —NCH).

FAB-MS m/z 184.3 (⁺M+1)

Example 13 Synthesis of2-(N-acetylpiperidine-4′-spiro)-6,6-dimethylpiperidine-4-one andbis(N-acetylpiperidine-4′-spiro)-2,6-piperidine-4-one

A reaction was performed in a manner similar to that in Step A ofExample 2, except that 2.12 g of N-acetyl-4-piperidone was used insteadof cyclohexanone. As a result,2-(N-acetylpiperidine-4′-spiro)-6,6-dimethylpiperidine-4-one andbis(N-acetylpiperidine-4′-spiro)-2,6-piperidine-4-one were obtained. Asan extraction solvent, chloroform was used. Oily substance was separatedusing silica gel column chromatography (developing solvent; 1 to 5%methanol: CHCl₃), thereby obtaining 556 mg of2-(N-acetylpiperidine-4′-spiro)-6,6-dimethylpiperidine-4-one (crystalobtained by recrystallization from hexane-ethyl acetate; melting point:82 to 83° C.) and 328 mg ofbis(N-acetylpiperidine-4′-spiro)-2,6-piperidine-4-one (crystal obtainedby recrystallization from hexane-ethyl acetate; melting point: 165 to166° C.).

2-(N-acetylpiperidine-4′-spiro)-6,6-dimethylpiperidine-4-one

1H-NMR (CDCl₃) 1.18 (3H), 1.22 (3H), 1.51˜1.69 (4H), 2.04 (3H), 2.71(2H), 2.29 (2H), 3.35˜3.78 (4H).

FAB-MS m/z 239.2 (+M+1)

Bis(N-acetylpiperidine-4′-spiro)-2,6-piperidine-4-one

1H-NMR (CDCl₃) 1.59˜1.61 (8H+H₂O), 2.08 (6H), 2.38˜2.39 (4H), 3.37˜3.41(8H).

FAB-MS m/z 322.3 (+M+1)

Example 14 Synthesis ofbis(N-acetylpiperidine-4′-spiro)-2,6-piperidine-4-one-1-oxyl

A reaction was performed in a manner similar to that in Step B ofExample 1 using 110 mg ofbis(N-acetylpiperidine-4′-spiro)-2,6-piperidine-4-one. As a result, 95mg of bis(N-acetylpiperidine-4′-spiro)-2,6-piperidine-4-one-1-oxyl wasobtained.

Melting point: 162 to 163° C.

FAB-MS m/z 337.3 (+M)

Example 15 Synthesis of2-(17′β-hydroxy-17′α-methyl-5′α-androstane-3-spiro)-6,6-dimethylpiperidine-4-oneandbis(17′β-hydroxy-17′α-methyl-5′α-androstane-3′-spiro)-2,6-piperidine-4-one

A reaction was performed in a manner similar to that in Step A ofExample 2, except that 2.25 g of17′β-hydroxy-17′α-methyl-5′α-androstane-3-one was used instead ofcyclohexanone. As a result,2-(17′β-hydroxy-17′α-methyl-5′α-androstane-3-spiro)-6,6-dimethylpiperidine-4-oneandbis(17′β-hydroxy-17′α-methyl-5′α-androstane-3′-spiro)-2,6-piperidine-4-onewere obtained. As an extraction solvent, chloroform was used. Oilysubstance was separated using silica gel column chromatography(developing solvent; ethyl acetate:hexane), thereby obtaining 130 mg of2-(17′β-hydroxy-17′α-methyl-5′α-androstane-3-spiro)-6,6-dimethylpiperidine-4-one(crystal obtained by recrystallization from toluene; melting point:156.7 to 158.9° C.) and 210 mg ofbis(17′β-hydroxy-17′α-methyl-5′α-androstane-3′-spiro)-2,6-piperidine-4-one(crystal obtained by recrystallization from toluene; melting point:292.7° C.).

2-(17′β-hydroxy-17′α-methyl-5′α-androstane-3-spiro)-6,6-dimethylpiperidine-4-one

FAB-MS m/z 402.4 (+M+1)

1H-NMR (CDCl₃) 0.77 (Me), 0.84 (Me), 1.20 (Me), 1.20 (Me), 1.22 (Me),2.19 (2H), 2.26 (2H)

Bis(17′β-hydroxy-17′α-methyl-5′α-androstane-3′-spiro)-2,6-piperidine-4-one

FAB-MS m/z 648.0 (+M+1)

1H-NMR (CDCl₃) 0.81 (Me×2), 0.83 (Me×2), 1.21 (Me×2), 2.41 (2H)

Test Example Reactivities to Hydroxyl Radical and Reactivities toAscorbic Acid

In order to examine reactivities of the following 3 types of TEMPO-basednitroxyl radical derivatives, in which substituents at position-2 andposition-6 in one derivative are different from those in anotherderivative, to free radical:

according to the method of Takeshita et al. (Biochimica et BiophysicaActa, 1573, 156-164 (2002)), the following procedure was carried out.Nitroxyl radical (25 μM) was mixed with H₂O₂ (10 mM), and thereafter aspecific amount of the mixture was collected in a capillary andirradiated with UV (100 to 120 mW/cm²) to generate OH radical. Thereactivities thereof to the nitroxyl radical were examined using X-bandESR. As a result, the 3 types of nitroxyl radical derivatives exhibitedthe same level of reactivity to OH radical (see FIG. 1). Thereactivities to hydroxyl radical were as follows: Compound 1>Compound3>Compound 2.

It has been reported that nitroxyl radical also reacts with a reductionsubstance (Couet, W. R., Tetrahedron, 41(7), 1165-1172 (1985);Finkelstein, E., Biochimica et Biophysica Acta, 802, 90-98 (1984)).Therefore, the following procedure was carried out. After nitroxylradical (10 to 50 μM) was mixed with ascorbic acid (100 μM to 2 mM), aspecific amount of the mixture was collected in a capillary, andthereafter the measurement was started. Using X-band ESR, decrease inESR signal intensity was observed over time, thereby examining thereactivity to ascorbic acid, which is a typical reduction substance inthe living body. As a result, it was found that tetraethyl derivative(Compound 2) almost never reacts with ascorbic acid. Meanwhile, when thereactivity of 2,6-dispiro-1′,1″-dipyran-piperidone-1-oxyl (Compound 3)to ascorbic acid was examined, it was found that2,6-dispiro-1′,1″-dipyran-piperidone-1-oxyl very rapidly reacts withascorbic acid (see FIG. 2). The reactivities of the compounds toascorbic acid were as follows: Compound 3>Compound 2>Compound 1. Theresults indicate that the reactivity to OH radical or ascorbic acid ischanged by changing substituents at position-2 and position-6 in theTEMPO-based compound.

The above-described results indicate that a compound in which position-2and position-6 in the TEMPO-based compound are substituted can be easilysynthesized by employing the novel synthesis pathway of the presentinvention. According to the production method of the present invention,it is possible to create a compound which can control the reactivity toa free radical or antioxidative substance.

INDUSTRIAL APPLICABILITY

The nitroxyl radical derivative obtained according to the presentinvention can be widely used as a contrast agent, an antioxidativeagent, etc. as well as a cell, a polymerization agent, etc. in the fieldof chemical industry. In particular, the ¹⁵N-labeled compound ofnitroxyl radical is useful as a contrast agent for following a freeradical reaction in vivo.

1. A method for producing a 2,6-substituted-4-piperidone derivativerepresented by the following formula:

wherein in the formula: R¹, R², R³ and R⁴ are each independently ahydrogen atom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenyl group, a C₂-C₂₀alkynyl group, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ aryl group, a C₆-C₂₀alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀ cycloalkyl group, aC₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl) C₁-C₁₀ alkyl group,and the aforementioned groups may be interrupted by an oxygen atom, anitrogen atom or a sulfur atom; any one of R¹, R², R³ and R⁴ is a groupother than a hydrogen atom; and R¹ and R² and/or R³ and R⁴ may beindependently crosslinked to each other to form a substituted orunsubstituted C₄-C₄₀ monocyclic or polycyclic saturated ring, and thesaturated ring may be interrupted by an oxygen atom, a nitrogen atom ora sulfur atom, wherein the method comprises the step of reacting atriacetoneamine derivative represented by formula (1):

wherein in the formula: R is a hydrogen atom or a C₁-C₆ alkyl group; andthe R′ groups are each independently a C₁-C₆ alkyl group, with theproviso that when the triacetoneamine derivative represented by theformula (1) is completely identical to the 2,6-substituted-4-piperidonederivative represented by the formula (1), it is excluded, with a ketoneor aldehyde derivative represented by formula (2):

wherein in the formula: R⁵ and R⁶ are each independently a hydrogenatom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenyl group, a C₂-C₂₀ alkynylgroup, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ aryl group, a C₆-C₂₀alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀ cycloalkyl group, aC₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl) C₁-C₁₀ alkyl group,and the aforementioned groups may be interrupted by an oxygen atom, anitrogen atom or a sulfur atom; any one of R⁵ and R⁶ is a group otherthan a hydrogen atom; and R⁵ and R⁶ may be crosslinked to each other toform a substituted or unsubstituted C₄-C₄₀ monocyclic or polycyclicsaturated ring, and the saturated ring may be interrupted by an oxygenatom, a nitrogen atom or a sulfur atom, in the presence of ammoniumsalt.
 2. The production method according to claim 1, wherein in theformula (1), R is a methyl group.
 3. The production method according toclaim 1, wherein the 2,6-substituted-4-piperidone represented by theformula (I) and the ammonium salt are ¹⁵N-labeled compounds.
 4. A2,6-substituted-4-piperidone derivative, which is obtained using themethod according to claim
 1. 5. A 2,6-substituted-4-piperidonederivative represented by formula (1′):

wherein in the formula: R^(1′), R^(2′), R^(3′) and R^(4′) are eachindependently a hydrogen atom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenylgroup, a C₂-C₂₀ alkynyl group, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ arylgroup, a C₆-C₂₀ alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀cycloalkyl group, a C₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl)C₁-C₁₀ alkyl group, and the aforementioned groups may be interrupted byan oxygen atom, a nitrogen atom or a sulfur atom; any one of R^(1′),R^(2′), R^(3′) and R^(4′) is a group other than a hydrogen atom; andR^(1′) and R^(2′) and/or R^(3′) and R^(4′) are independently crosslinkedto each other to form a substituted or unsubstituted C₄-C₄₀ monocyclicor polycyclic saturated ring, and the saturated ring may be interruptedby an oxygen atom, a nitrogen atom or a sulfur atom, with the provisothat R^(1′) and R^(2′) and/or R^(3′) and R^(4′) do not form acyclohexane ring with no substituent.
 6. The2,6-substituted-4-piperidone derivative according to claim 5, which isrepresented by formula (3):

wherein in the formula: X¹ and X² are each independently a substitutedor unsubstituted C₄-C₂₀ cycloalkylene group which may be interrupted byan oxygen atom, a nitrogen atom or a sulfur atom; carbonyl group;acetamide group; sulfonyl group; sulfinyl group; an oxygen atom; or asulfur atom.
 7. A 2,6-substituted-4-piperidone derivative representedby:


8. The 2,6-substituted-4-piperidone derivative according claim 7,wherein a nitrogen nucleus is labeled with ¹⁵N.
 9. A method forproducing a nitroxyl radical derivative represented by formula (II):

wherein in the formula: R¹, R², R³ and R⁴ are each independently ahydrogen atom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenyl group, a C₂-C₂₀alkynyl group, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ aryl group, a C₆-C₂₀alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀ cycloalkyl group, aC₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl) C₁-C₁₀ alkyl group,and the aforementioned groups may be interrupted by an oxygen atom, anitrogen atom or a sulfur atom; any one of R¹, R², R³ and R⁴ is a groupother than a hydrogen atom; and R¹ and R² and/or R³ and R⁴ may beindependently crosslinked to each other to form a substituted orunsubstituted C₄-C₄₀ monocyclic or polycyclic saturated ring, and thesaturated ring may be interrupted by an oxygen atom, a nitrogen atom ora sulfur atom, wherein the method comprises the step of producing anitroxyl radical by oxidizing an amino group of a2,6-substituted-4-piperidone derivative obtained using the methodaccording to claim 1, which is represented by the following formula:

wherein in the formula: R¹, R², R³ and R⁴ mean the same as describedabove.
 10. The production method according to claim 9, wherein thenitroxyl radical derivative represented by the formula (II), theammonium salt and the 2,6-substituted-4-piperidone derivativerepresented by the formula (I) are ¹⁵N-labeled compounds.
 11. A nitroxylradical derivative, which is obtained using the method according toclaim
 9. 12. A nitroxyl radical derivative represented by formula (II′):

wherein in the formula: R^(1′), R^(2′), R^(3′) and R^(4′) are eachindependently a hydrogen atom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenylgroup, a C₂-C₂₀ alkynyl group, a C₄-C₂₀ alkyldienyl group, a C₆-C₁₈ arylgroup, a C₆-C₂₀ alkylaryl group, a C₆-C₂₀ arylalkyl group, a C₄-C₂₀cycloalkyl group, a C₄-C₂₀ cycloalkenyl group, or a (C₃-C₁₀ cycloalkyl)C₁-C₁₀ alkyl group, and the aforementioned groups may be interrupted byan oxygen atom, a nitrogen atom or a sulfur atom; any one of R^(1′),R^(2′), R^(3′) and R^(4′) is a group other than a hydrogen atom; andR^(1′) and R^(2′) and/or R^(3′) and R^(4′) are independently crosslinkedto each other to form a substituted or unsubstituted C₄-C₄₀ monocyclicor polycyclic saturated ring, and the saturated ring may be interruptedby an oxygen atom, a nitrogen atom or a sulfur atom, with the provisothat R^(1′) and R^(2′) and/or R^(3′) and R^(4′) do not form acyclohexane ring with no substituent.
 13. The nitroxyl radicalderivative according to claim 12, which is represented by formula (4):

wherein in the formula: X¹ and X² are each independently a substitutedor unsubstituted C₄-C₂₀ cycloalkylene group which may be interrupted byan oxygen atom, a nitrogen atom or a sulfur atom; carbonyl group;acetamide group; sulfonyl group; sulfinyl group; an oxygen atom; or asulfur atom.
 14. A nitroxyl radical derivative represented by:


15. The nitroxyl radical derivative according to claim 14, wherein anitrogen nucleus is labeled with ¹⁵N.